本文選題：南極苔蘚 + 紫外輻射��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：南極洲位于地球南端,四周被三大洋包圍,與其他大陸隔離,大陸面積約1240萬平方千米,占地球陸地總面積的十分之一,但僅分布850多種植物,且絕大部分為低等植物。強(qiáng)紫外是制約南極陸生植物生長(zhǎng)發(fā)育的主要因素之一。苔蘚植物(Bryophytes)是南極大陸分布最廣、數(shù)量最多的綠色植物。苔蘚適應(yīng)極端環(huán)境強(qiáng)輻射的機(jī)制與其特殊的結(jié)構(gòu)基因及代謝途徑密切相關(guān)。類黃酮化合物(Flavonoids)是植物體內(nèi)最大的一類多羥基酚類次生代謝產(chǎn)物,從高等植物到較低等的植物類群都有分布,在植物適應(yīng)紫外輻射過程中起著重要作用。在高等植物中,類黃酮代謝途徑較為清晰,其起始于苯丙烷類代謝途徑,經(jīng)查爾酮合酶和查爾酮異構(gòu)酶催化生成類黃酮化合物的前體物質(zhì)柚皮素(黃烷酮);柚皮素作為類黃酮代謝途徑的前體化合物,被黃烷酮3-羥化酶、黃酮醇合成酶、花青素合成酶和黃酮合酶等酶催化進(jìn)入不同的分支代謝途徑。然而低等植物的類黃酮代謝途徑仍不清楚,目前僅有小立碗蘚查爾酮合酶家族及查爾酮異構(gòu)酶,和一個(gè)苔類的黃酮合酶的報(bào)道。本論文分析了紫外輻射下,南極絲瓜蘚類黃酮化合物的代謝變化;克隆了類黃酮代謝途徑的關(guān)鍵酶查爾酮合酶家族基因和一個(gè)黃酮合酶基因(Flavone synthase,FNS),分析了其對(duì)非生物脅迫的響應(yīng)特征,研究了FNS重組蛋白的體外的催化活性和在擬南芥中的體內(nèi)催化活性。具體結(jié)果如下:1南極苔蘚紫外輻照條件下黃酮類物質(zhì)的響應(yīng)利用UV-B輻射處理南極苔蘚(絲瓜蘚屬)和常溫苔蘚(絲瓜蘚屬,來自山東省),經(jīng)UV-B輻射處理100 h后,結(jié)果發(fā)現(xiàn)常溫苔蘚莖葉體上端枯死,當(dāng)在黑暗條件下恢復(fù)12 h后,枯死程度繼續(xù)加劇;而南極苔蘚的擬莖由嫩綠色變?yōu)樯詈稚?恢復(fù)生長(zhǎng)后擬莖變?yōu)闇\棕色。初步表明,與常溫苔蘚比,南極苔蘚對(duì)UV-B輻射的抗性更強(qiáng),這種抗性可能與多酚類化合物有關(guān)。利用HPLC研究了 UV-B輻射處理對(duì)南極絲瓜蘚類黃酮化合物代謝譜的影響。結(jié)果發(fā)現(xiàn),UV-B處理不僅增加了類黃酮化合物的含量,還誘導(dǎo)新的化合物的合成。2南極苔蘚查爾酮合酶家族基因的特征分析查爾酮合酶(Chalcone synthase,CHS)是植物類黃酮生物合成途徑中的第一個(gè)關(guān)鍵酶,在植物抵御外界環(huán)境脅迫中發(fā)揮重要作用。利用RACE技術(shù)從南極絲瓜蘚cDNA中獲取了 6條開放閱讀框約1200bp大小片段的查爾酮合酶家族基因(Pn021、Pn444、Pn768、Pn088、Pn847 和 Pn270)。序列比對(duì)分析發(fā)現(xiàn),PnCHS與來自其它物種的查爾酮合酶的蛋白序列相似度在24.0%-84.0%之間;Pn444、Pn088和Pn768具有保守的Cys164、His303和Asn336(氨基酸序號(hào)以苜蓿蛋白標(biāo)出)催化三聯(lián)體及GFGPG基序和兩個(gè)保守的與底物結(jié)合的口袋。利用近鄰比對(duì)法構(gòu)建PnCHS的系統(tǒng)進(jìn)化樹,結(jié)果顯示苔蘚植物的CHS聚類到同一分枝,位于菌類和高等植物的中間。構(gòu)建35S::Pn444::GFP/pBI221瞬時(shí)表達(dá)載體,利用PEG介導(dǎo)法轉(zhuǎn)化擬南芥原生質(zhì)體,熒光共聚焦顯微鏡下觀察綠色熒光蛋白信號(hào),發(fā)現(xiàn)目的蛋白主要分布于細(xì)胞內(nèi)膜性細(xì)胞器。利用qRT-PCR技術(shù)分析了不同脅迫處理下,PnCHS家族基因的響應(yīng)情況。結(jié)果表明,UV-B輻射下,PnCHS家族基因的表達(dá)水平均上調(diào)表達(dá);低溫處理下PnCHS表達(dá)水平都下調(diào)表達(dá);高鹽處理后南極苔蘚查爾酮合酶基因Pn021、Pn088和Pn444的表達(dá)水平下調(diào),而Pn270和Pn847的表達(dá)水平上調(diào);干旱脅迫后,Pn021、Pn444和Pn847的表達(dá)水平顯著上調(diào);MeJA和ABA處理后,Pn444和Pn847的表達(dá)水平顯著增加。3南極苔蘚黃酮合酶的體內(nèi)外酶活功能分析黃酮合酶(Flavone synthase)是類黃酮代謝途徑中重要的雙加氧酶之一,催化黃烷酮在C環(huán)2-3位形成雙鍵。通過篩選南極苔蘚轉(zhuǎn)錄組數(shù)據(jù)庫,克隆得到一個(gè)和黃酮合酶同源性較高的基因(PnFNS),與其他20DD家族相似性為20.0-80.0%,包含HXDX～50HK～10RXS motif、Fe2+結(jié)合位點(diǎn)、2-酮戊二酸結(jié)合位點(diǎn)以及柚皮素催化位點(diǎn)。通過構(gòu)建系統(tǒng)進(jìn)化樹,我們發(fā)現(xiàn)PnFNS與小立碗蘚中的相應(yīng)同源蛋白類聚在同一分枝;以擬南芥花青素合酶(2brt.pdb)晶體結(jié)構(gòu)為模板,利用SWISS-MODEL在線對(duì)PnFNS蛋白進(jìn)行同源建模,預(yù)測(cè)其催化機(jī)制。通過構(gòu)建熒光蛋白融合載體35S::PnFNS::GFP/pBI221轉(zhuǎn)入擬南芥原生質(zhì)體,觀察顯示熒光蛋白位于膜性細(xì)胞器,表明PnFNS可能主要定位在細(xì)胞膜和內(nèi)質(zhì)網(wǎng)。構(gòu)建PnFNS-pET28a進(jìn)行異源表達(dá),體外實(shí)驗(yàn)發(fā)現(xiàn),重組蛋白僅能催化柚皮素生成芹菜素,對(duì)二氫槲皮素、芹菜素、山奈酚、槲皮素均不發(fā)生反應(yīng)。通過pH3.0-11.0的緩沖液分析PnFNS的最適pH,結(jié)果發(fā)現(xiàn),PnFNS在低于pH4.0的反應(yīng)液內(nèi)催化活性很低,pH4.0-7.0之間酶活性隨著pH的升高而增加,大于pH7.0后酶活性又迅速下降。以pH7.0為最適pH進(jìn)行梯度溫度反應(yīng),發(fā)現(xiàn)PnFNS對(duì)低溫不敏感,在0-20℃期間均有較高活性;高于30℃時(shí),酶活性迅速下降。在pH7.0和15℃條件下,研究了不同離子對(duì)PnFNS的催化活性;結(jié)果表明,Mg2+、Ca2+、Mn2+和Cu2+對(duì)酶活性有提升作用,Co2+對(duì)酶活性有部分抑制作用,添加離子螯合劑Na2EDTA后完全抑制了酶活性,表明PnFNS是金屬離子依賴型低溫酶。將3 mg的野生型擬南芥和過表達(dá)PnFNS的轉(zhuǎn)基因擬南芥(AtOE)種子放入加有0或200 μM的柚皮素的液體1/2MS中靜止培養(yǎng)10d。結(jié)果發(fā)現(xiàn),正常培養(yǎng)情況下,野生型和轉(zhuǎn)基因擬南芥的生長(zhǎng)沒有差別,但是含有200 μM柚皮素的1/2MS中,過表達(dá)PnFNS在很大程度上解除了柚皮素對(duì)植株生長(zhǎng)的抑制,轉(zhuǎn)基因擬南芥的長(zhǎng)勢(shì)顯著強(qiáng)于野生型擬南芥。利用HPLC分析植物類黃酮代謝譜,發(fā)現(xiàn)柚皮素在野生型和轉(zhuǎn)基因擬南芥中都沒有明顯的積累,但是野生型的擬南芥中積累一個(gè)保留時(shí)間早于柚皮素的物質(zhì)(推測(cè)為:6-丙�；�-α-D-葡萄糖基-(1→7)-橙皮素),而轉(zhuǎn)基因擬南芥中積累了較多的芹菜素。表明南極苔蘚PnFNS在擬南芥體內(nèi)具有和體外類似的催化反應(yīng)。將3周齡的野生型擬南芥和過表達(dá)PnFNS的擬南芥在添加UV-B(20 μW/um2)的全波長(zhǎng)光源下處理6h,然后恢復(fù)生長(zhǎng)2d,觀察葉片損傷情況并通過DAB染色檢測(cè)葉片活性氧的含量。結(jié)果發(fā)現(xiàn),過表達(dá)擬南芥葉片壞死面積和活性氧水平均明顯少于野生型,表明過表達(dá)PnFNS擬南芥增強(qiáng)了植物對(duì)紫外的耐受能力。
[Abstract]:Antarctica is located at the southern end of the earth, surrounded by three oceans and isolated from other continents. The continent is about 12 million 400 thousand square kilometers, accounting for 1/10 of the total area of the earth's land, but only 850 kinds of plants are distributed, and most of them are low plants. The strong ultraviolet (Bryophy) is one of the main factors restricting the growth and development of Antarctic Lu Shengzhi. Bryophytes (bryophyte) TES) is the most widely distributed and largest green plant in the Antarctic continent. The mechanism of moss adapted to extreme environmental strong radiation is closely related to its special structural genes and metabolic pathways. The flavonoid compound (Flavonoids) is the largest secondary metabolite of polyhydroxyphenols in plants, from higher plants to lower plant groups. Distribution, plays an important role in the process of plant adaptation to ultraviolet radiation. In higher plants, the metabolic pathway of flavonoids is clearer. It begins in the phenylpropanoid pathway and catalyzes the production of the precursor of the flavonoids by chalcone synthase and chalcone isomerase in the precursor of naringin (Huang Wantong); naringenin is a precursor to the metabolic pathway of flavonoids. Body compounds, which are catalyzed by flavanone 3- hydroxylase, Flavonol Synthase, anthocyanin synthetase, and flavonoid synthase, are catalyzed into different branch metabolic pathways. However, the metabolic pathways of flavonoids in lower plants are still unclear. Currently, only the chalcone synthase family and the chalcone isomerase, and the flavonoid synthase of a liverwort are reported in the lower plants. In this paper, the metabolic changes of flavonoids in the Antarctic gourd moss were analyzed under ultraviolet radiation, and the key enzymes of the flavonoid metabolism pathway, chalcone synthase family gene and a flavonoid synthase gene (Flavone synthase, FNS), were cloned, and their response to abiotic stress was analyzed, and the catalytic activity of FNS recombinant protein in vitro was studied. The specific results are as follows: 1 the response of the flavonoids in the UV irradiated Antarctic moss under the UV irradiation conditions by UV-B radiation treatment of the moss of Antarctic (MOSS MOSS) and the moss of normal temperature (from Shandong province) and after 100 h treatment by UV-B radiation, the fruit found that the upper end of the stem and leaf of the moss at normal temperature was dead, when in the dark strip. After the recovery of 12 h, the death degree continued to intensify, and the pseudo stem of the Antarctic moss changed from tender green to dark brown, and the pseudo stem became light brown after the recovery. It was preliminarily indicated that the resistance of Antarctic moss to UV-B radiation was stronger than that of moss at normal temperature. This resistance may be related to polyphenols. The use of HPLC to study the treatment of UV-B radiation to Antarctica. The results showed that the UV-B treatment not only increased the content of flavonoids, but also induced the synthesis of new compounds,.2, the characteristics of the gene of chalcone synthase in the moss Antarctica, the Chalcone synthase (CHS) was the first key enzyme in the biosynthesis pathway of the plant flavonoids. The RACE technique was used to obtain 6 chalcone synthase family genes (Pn021, Pn444, Pn768, Pn088, Pn847 and Pn270) from the Antarctic gourd moss cDNA by using RACE technology. The sequence alignment analysis found that PnCHS and the protein of chalcone synthase from other species Sequence similarity is between 24.0%-84.0%; Pn444, Pn088 and Pn768 have the conservative Cys164, His303 and Asn336 (amino acid sequence number with alfalfa protein) to catalyze the three body and GFGPG motif and two conservative pockets with the substrate. The phylogenetic tree of PnCHS is constructed by near neighbor comparison method. The results show that the CHS cluster of the moss plants to the same one. Branches, located in the middle of fungi and higher plants. Construct 35S:: Pn444:: GFP/pBI221 transient expression vector, transform Arabidopsis Protoplast by PEG mediated method, observe green fluorescent protein signal under fluorescence confocal microscope, find that the target protein is mainly distributed in cell endocardium cell organelles. The different stress treatment is analyzed by qRT-PCR technology. The response of PnCHS family genes showed that the expression level of PnCHS gene expression was up regulated under UV-B radiation; PnCHS expression level was down regulated under low temperature treatment; the expression level of Pn021, Pn088 and Pn444 in Antarctic lichen chalcone synthase gene was down regulated, and the expression level of Pn270 and Pn847 was up-regulated after high salt treatment; drought stress was up. After the treatment of Pn021, Pn444 and Pn847, the expression level of MeJA and ABA significantly increased the expression level of Pn444 and Pn847 in.3 Antarctic moss flavonoid synthase activity analysis of the flavonoid synthase (Flavone synthase), one of the most important bioxygenase in the flavonoid metabolic pathway, catalyzing the formation of a double bond in the 2-3 position of the flavanone in the C ring. Screening the database of the Antarctic moss transcriptional group to clone a gene (PnFNS) with a high homology of the flavonoid synthase, which is similar to that of other 20DD families, including HXDX to 50HK to 10RXS motif, Fe2+ binding site, 2- ketone glutaric acid binding site and naringin catalytic site. By constructing phylogenetic tree, we found PnFNS and small. The corresponding homologous proteins in the erect moss were clustered in the same branch; using the Arabidopsis anthocyanin synthase (2brt.pdb) crystal structure as a template, the homologous modeling of PnFNS protein was established by SWISS-MODEL online, and its catalytic mechanism was predicted. The fluorescent protein fusion carrier, 35S:: PnFNS:: GFP/pBI221, was transferred into the Arabidopsis protoplast, and the fluorescent eggs were observed and displayed. It is located in the membranous organelle, indicating that PnFNS may be mainly located in the cell membrane and endoplasmic reticulum. PnFNS-pET28a is constructed for the heterologous expression. In vitro, the recombinant protein can only catalyze the production of naringin, and it does not respond to two hydrogen quercetin, apigenin, kaempferol and quercetin, and the optimum analysis of PnFNS by pH3.0-11.0 buffer solution. PH, the results showed that the catalytic activity of PnFNS in the reaction liquid below pH4.0 was very low, the activity of pH4.0-7.0 increased with the increase of pH, and the activity of the enzyme decreased rapidly after the pH7.0. PH7.0 was the optimum pH for the gradient temperature reaction. It was found that PnFNS was insensitive to low temperature and had high activity at 0-20 degrees C, and the enzyme activity was rapid when higher than 30. Down. The catalytic activity of different ions on PnFNS was studied at pH7.0 and 15 C. The results showed that Mg2+, Ca2+, Mn2+ and Cu2+ had a promotion of enzyme activity, Co2+ had partial inhibition on the activity of enzyme, and the activity of the enzyme was completely suppressed after the addition of the ion chelating agent Na2EDTA. It was indicated that PnFNS was a metal ion dependent cryogenic enzyme. The wild type of 3 mg was found. In Arabidopsis and PnFNS transgenic Arabidopsis (AtOE) seeds in the liquid 1/2MS with 0 or 200 mu M in the liquid 1/2MS, it was found that there was no difference in the growth of wild type and transgenic Arabidopsis in normal culture, but the overexpression of PnFNS in 1/ 2MS containing 200 u M naringin greatly relieved the grapefruit. The growth of transgenic Arabidopsis was significantly stronger than that of wild Arabidopsis. Using HPLC to analyze the metabolic profiles of flavonoids in plants, it was found that naringin had no obvious accumulation in wild type and transgenic Arabidopsis, but in wild Arabidopsis, the accumulation time was earlier than that of naringenin (presumably 6-). The propanoyl - alpha -D- glucosamine - (1 - 7) - hesperidin, and the accumulation of more apigenin in transgenic Arabidopsis. It shows that the Antarctic moss PnFNS has a similar catalytic reaction in the body with the Arabidopsis in vitro. The 3 week old wild Arabidopsis and the Arabidopsis thaliana overexpressing PnFNS are treated with the full wavelength light source of UV-B (20 mu) with the full wavelength light source, and then the Arabidopsis thaliana is treated with the whole wavelength light source. The growth of 2D was restored and the leaf damage was observed and the content of active oxygen in leaves was detected by DAB staining. The results showed that over expression of the leaf necrosis area and active oxygen water of Arabidopsis thaliana was significantly less than that of the wild type, indicating that over expression of PnFNS Arabidopsis enhanced the tolerance of plants to ultraviolet.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2

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